The use of polymeric particles and magnetic particles to bind a compound has long been known and used in industrial and laboratory procedures. For example, the Merrifield resins, crosslinked styrene-divinylbenzene spheroidal beads, were among the earliest and most widely used modern substrate particles. They were used in organic synthesis, for heterogenizing homogeneous catalysts and in biochemical reactions. Since the Merrifield resins were fairly large, they could easily be separated by filtration. In some fields, however, it is desirable to use colloidal sized particles because the material to be bound is scarce, expensive or is to be used in a procedure where larger particles are not desirable. This is particularly true in the biochemical field. When particles are of colloidal size, however, their separation from liquid medium by filtration can become lengthy and difficult. In particular, colloidal particles tend to coat the surface of the filter and slow the process. The use of magnetic particles, specifically magnetic particles having a polymeric coating, has found great utility because such particles can be magnetically gathered to one side of a reaction vessel and the bulk of the reaction medium simply decanted. (The word "particles" as used herein encompasses spheres, spheroids, beads and other shapes as well. These words are used interchangeably unless otherwise specified.) The use of coated magnetic particles has found a particular utility in biological applications, especially where antibodies are bound to the surface coating of the particles. The bound antibodies may then be used to capture a specific biological substance from a test sample containing numerous biological samples or to capture undesired species from the test sample, leaving the desired species in the sample.
The categories of coated magnetic particles, also known as magnetic spheres or beads, can be divided into four general classes.
1. Core-and-shell beads with a magnetic core and a hard shell coating of polymerized monomer or a silanizing agent. See U.S. Pat. No. 4,267,234 to Rembaum (polyglutaraldehyde shell around ferrofluid core particles); U.S. Pat. No. 4,454,234 to Czerlinski (suspension or emulsion polymerized coating around submicron magnetic particles); U.S. Pat. Nos. 4,554,088, 4,695,392 and 4,695,393 to Whitehead et al. (silanized magnetic oxide particles of polydisperse size and shape); U.S. Pat. No. 4,672,040 to Josephson (polysilane coated magnetic particles); U.S. Pat. No. 4,783,336 to Margel et al. (suspension polymerized polyacrolein around ferrofluid particles); U.S. Pat. No. 4,795,698 to Owen et al. (bovine serum albumin coating); and U.S. Pat. No. 4,964,007 to Yudelson (gelatin-gum arabic-surfactant coating); PA1 2. Core-and-shell beads with a magnetic core and a loose shell of random coil or globular polymer which may or may not be crosslinked. See U.S. Pat. No. 4,452,773 to Molday (dextran coating around ferrofluid particles) and U.S. Pat. No. 4,795,698 to Owen et al. (protein such as bovine serum albumin around ferrofluid particles. PA1 3. Magnetic latex materials formed by uniformly embedding ferrofluid particles in polystyrene latex particles. See U.S. Pat. No. 4,358,388 to Daniel et al. PA1 4. Porous polymer particles filled with magnetic materials such as polymer-ferrite or polymer maghemite composite systems. See K. Nustad et al. "Monodisperse Polymer Particles In Immunoassays And Cell Separation", Microspheres: Medical and Biological Applications, A. Rembaum and Z. Tokes, eds. (Boca Raton, Fla.: CRC Press, 1988) pages 53-75; C.D. Platsoucas et al., "The Use Of Magnetic Monosized Polymer Particles For The Removal Of T Cells From Human Bone Marrow Cell Suspensions", ibid. at pages 89-99; and U.S. Pat. Nos. 4,563,510, 4,530,956 and 4,654,267 [International Patent Publication No. WO 83/03920] to Ughelstad et al. (polymer coated magnetic particles prepared by treating compact or porous particles with a solution of iron salts and the use of such particles for medical, diagnostic or other purposes). PA1 1. The particles should be as small as possible in order to maximize the surface area on which the biological reagent is coated, but the particles should still be easily separable with a small magnet. Small size and large surface area are desirable in order to use the least possible quantity of particles to remove the targeted substance; e.g., to interact with on the order of 10.sup.6 cells per sample in one step, thereby avoiding sequential additions and work-ups. PA1 2. There should be a low non-specific binding of the antibody-coated particles to cell surfaces. The particle surface should be hydrophilic or covered with a coating of a hydrophilic substance to which the antibody is attached. PA1 3. The polymer and antibody layers on the particles should be covalently bound to each other in order to reduce dissociation and conformational changes. PA1 4. The coating on the magnetic particles and any molecular chains which link an antibody to the polymer surface should be metabolizable. PA1 5. In positive selection of cells, a mechanism for quickly and easily recovering viable cells from the magnetic particles should be available in order that recovered cells can be cultured. PA1 6. In the negative selection of cells, the antibody-coated particles should be sterile so that the remaining cells can be cultured. PA1 7. For magnetic separation and sorting of cells and other biological substances, the preferred magnetic particles are "soft" magnetic particles. That is, particles which can be easily magnetized and demagnetized as opposed to hard or permanent magnetic. The particles can be ferromagnetic, ferrimagnetic or superparamagnetic. Ferromagnetic and ferrimagnetic particles are not limited in size, whereas superparamagnetic particles are limited to single domain structures of dimensions usually less than about 40 nanometers. (C. Kittel et al., Solid State Physics 3: 437-464 (1956). PA1 1. Ferrofluid core and the usual polymer outer shell particles have too small a magnetic moment to make them practical for use in cell separations where hand-held permanent magnets are used to collect and separate the magnetic particles. Such particles require the use high-field separation techniques which severely limits the volume of material which can be processed, thus limiting scale-up. PA1 2. Ferrofluid-polystyrene particles prepared by emulsion polymerization cannot be tightly controlled in size and range from about 0.1 to 4 .mu.m in diameter. Consequently, in cell separations using antibodies conjugated to such beads, the very small, kinetically-mobile magnetic particles which inherently possess the least magnetic moment tend to preferentially occupy the antigenic sites on a cell surface. As a result, the resulting cell-bead conjugates do not have a sufficient magnetic moment to permit easy separation.
The usefulness of most polymer coated magnetic beads in medical and biological applications has been limited by practical considerations such as the uniformity of particle size and shape, the need for the biological reagent to be strongly bound to the particle, a preference for hydrophilic polymer coatings as opposed to hydrophobic coatings, and whether or not the coating is biodegradable. While biodegradability is of particular importance where a biological reagent is to be administered in vivo, it is also important in various cell sorting, separation and assay procedures. The most desirable coated magnetic particles would have the following features.
Problems exist with using each of the magnetic-composite particles from each of the above class in cell separation procedures. Some examples of the problems encountered are:
The use of magnetic particles having first and second layers of types B and A gelatin, respectively, and prepared as taught herein and in the priority application Ser. No. 07/607,253, now U.S. Pat. No. 5,169,754, issued Dec. 8,1992 overcomes these difficulties. However, gelatin coated particles have been found to have some problems regarding non-specific interactions with certain cells, notably platelets and phagocyte cells such as monocytes. The problem arises because the amino acid sequence of gelatin (as exemplified by the .alpha.-1 chain of rat and calf skin collagen) includes three regions with the tripeptide sequence Arg-Gly-Asp (RGD) which duplicates the RGD binding sequence of fibronectin, a component of the extracellular matrix that specifically promotes cellular adhesion. Those biological cells with fibronectin expressed on their surface have a specific affinity for collagen, which is equivalent to crosslinked gelatin. For example, antibody containing gelatin coated magnetic ferrite particles used in the separation of subsets of white blood cells will also bind to fibronectin on the surface of platelets and monocytes. The result is non-specific depletion of cells because monocytes and platelets are bound to the particles as well as those cells which bear antibody-specific antigens. The non-specific depletion of cells can be avoided through the use of an aminodextran as the outermost coating layer on coated particles. The use of dextran derivatives as carriers has been discussed by U. Manabe et al., J. Lab. Clin. Med. 104: 445-454 (1984) (antibody-polyaldehyde dextran-methotrexate); L. B. Shin et al., Intl. J. Cancer 41: 832-839 (1988) (antibody-aminodextran-methotrexate); A. R. Oseroff et al., Proc. Natl. Acad. Sci. USA 83: 8744-8748 (1986) (antibody-aminodextran-chlorine 6); S. Rakestraw et al., Proc, Natl., Acad. Sci. USA 87: 4217-4221 (1990) (antibody-dextran hydrazide-Sn(IV) chlorine 6); R. J. Mrsnay et al., Eur. J. Cell. Biol. 45: 200-208 (1987) (ouabain-aminodextran-gold particle); J. W. M. Bulte et al., Magnetic Res. 25: 148-157 (1992) (anti particle); and other as described in S. S. Wong, "Chemistry of Protein Conjugation and Cross-Linking" (CRC Press, Boca Raton, Fla. 1991).
The various particles described above have been used in the biological arts to immobilize a variety of biological substances, particularly antibodies. In using such particles, immobilization of antibodies by covalent coupling is preferred to immobilization by antibody adsorption which requires careful and separate adjustment of pH and antibody concentration for each monoclonal antibody used. P. Bagchi et al., J. Colloid Interface Sci., 83: 460-478 (1981); J. Lyklema, Colloids and Surfaces, 10:33-42 (1984); M. D. Bale et al., J. Colloid Interface Sci., 125: 516-525 (1988); C. C. Ho et al., ibid., 121: 564-570 (1988); "Proteins at Interfaces: Physicochemical and Biochemical Studies", ACS Symposium Series, No. 343, J. L. Brash and T. A. Horbett, Eds. (Washington: Amer. Chem. Soc., 1987); W. Norde, Adv. Coll. Interface Sci., 25: 267-340 (1986); A. V. Elgersma et al., Abstracts of the 198th Amer. Chem. Soc. Meeting, Miami Beach, Fla., Sep. 10-15, 1989, COLL 0131; and D. E. Brooks, Annenberg Center for Health Sciences and H. B. Wallis Research Facility at Eisenhower Latex Conference, Orlando, Fla., Dec. 4-5, 1989. However, even when the pH and antibody are carefully controlled, there is little assurance that the orientation of adsorbed antibody will be such that an active adsorbed antibody will result. Adsorbed antibodies also have long term storage problems arising from antibody desorption from the particles' surfaces. Furthermore, proteins, such as antibodies, tend to achieve maximum adsorption on hydrophobic surfaces at or near the pI of the protein. However, if electrostatic interactions between charge groups are important, then the adsorbing surface and the adsorbate should have net opposite charges. Covalent coupling methods, on the other hand, are not as sensitive to these conditions.
Covalent coupling methods have been used with particles of magnetite embedded in carboxy-modified latex subsequently coated with aminodextran [R. S. Molday et al. FEBS. Lett., 170: 232-238 (1984)] and derivatized with a number of antibodies as described in application Ser. No. 07/255,743 (now abandoned), and in copending application Ser. No. 07/961,157 filed Oct. 15, 1992 and entitled POLYMERIC PARTICLES HAVING A BIODEGRADABLE GELATIN AND AMINODEXTRAN COATING AND PROCESS FOR MAKING SAME which is incorporated herein by reference. If the antibody is of IgG isotype, the covalent coupling method assures that the linkage between the antibody and the particles occurs at the antibody Fc or hinge region, and not at the antibody's Fab region. If the antibody is of pentameric IgM isotype which has only Fab regions exposed, the coupling of one Fab region to the particle will still leave four Fab regions exposed and available for reaction.
This invention provides for the preparation of magnetic particles having a biodegradable coating to which can be attached pendent biological substances, such as monoclonal antibodies. The particles of the invention can be used in various cell separation and assay methodologies. Biodegradability in the coating used on the magnetic core material is important in cell separation technology. For example, antibodies may be conjugated to gelatin/aminodextran coated magnetic particles such as manganese ferrite particles. These particles would contain a coating and a manganese-iron oxide core, all of which are biodegradable. In a positive cell selection procedure using such particles, once the desired cell has been isolated from other cells, the particles and coating can be allowed to degrade in a manner such that the cells are kept viable and can be cultured for further use. Alternatively, the enzyme collagenase can be used first to release the core material (magnetic or latex) by digestion of the gelatin/aminodextran coating. The core material can then be removed from the cell suspension before culturing the cells. In the negative selection of cells with such biodegradable beads, the beads can be left in the cell suspension from which targeted cells were removed without compromising the viability of the remaining cells. For example, in bone marrow purging operations using biodegradable magnetic beads, there is less concern about leaving behind some beads in the purged marrow that is to be transplanted in a patient. Currently, synthetic polymer-magnetite particles prepared by Ughelstad et al, U.S. Pat. No. 4,654,267 (WO 83/03920), and conjugated with antibody are being used in bone marrow purging. The polymer is not biodegradable and imparts a hydrophobic surface to these beads. This hydrophobicity, which is not present in the gelatin/aminodextran coated particles of the claimed invention, is responsible for non-specific interactions between the beads and cells. As a result of this non-specific interaction, the selectivity is poor and more beads must be used to attain the desired level of treatment. The claimed invention avoids these problems.